EP0126061A1

EP0126061A1 - Process for the treatment of a proteinic substance with a view to separating the proteins and the prosthetic groups, and applications to haemoglobin and chlorophyl

Info

Publication number: EP0126061A1
Application number: EP19820903321
Authority: EP
Inventors: Patrick Espenan
Original assignee: SODEPRAL SA
Current assignee: SODEPRAL SA
Priority date: 1982-11-09
Filing date: 1982-11-09
Publication date: 1984-11-28
Also published as: WO1984001883A1; AU9129582A

Abstract

Procédé de traitement d'une substance protéique en vue de séparer la ou les protéines de celle-ci du ou des groupements prosthétiques. Ce procédé consiste, dans une première étape, à disposer la substance protéique en milieu acide dilué de pH compris entre 0,5 et 5 et, dans une seconde étape, à séparer la phase solide du précipité formé pour conserver la phase liquide contenant préferentiellement en solution la ou les protéines de la substance. Le procédé de l'invention peut en particulier être appliqué à l'hémoglobine en vue de séparer la globine ou à la chlorophylle en vue de la séparation des protéines contenues dans celle-ci.Process for the treatment of a proteinaceous substance in order to separate the protein or proteins thereof from the prosthetic group or groups. This process consists, in a first step, in placing the protein substance in a dilute acidic medium with a pH of between 0.5 and 5 and, in a second step, in separating the solid phase from the precipitate formed in order to preserve the liquid phase preferably containing solution of the substance (s). The process of the invention can in particular be applied to hemoglobin in order to separate the globin or to chlorophyll in order to separate the proteins contained therein.

Description

PROCESS FOR TREATING A PROTEIN SUBSTANCE WITH A VIEW TO SEPARATING PROTEINS AND PROSTHETIC GROUPS, AND APPLICATIONS TO HEMOGLOBIN AND CHLOROPHYLLE

The invention relates to a method for treating a protein substance composed of at least one protein and at least one prosthetic group in order to separate said protein (s) from said prosthetic group (s); it extends in particular to applications with a view to separating substantially white proteins from colored prosthetic groups, for example separation of the globin from a substance containing hemoglobin (cruor, blood, hematia), separation of the proteins contained in chlorophyll or substances derived from or derived therefrom. It is known that proteins are one of the essential elements of foodstuffs and many products are enriched in proteins as well for the food of the man as for that of the animals. However the blood and particularly the cruor which is derived from it is very rich in hemoglobin composed of the association of heme (constituting a prosthetic group) and globin (constituting a white protein). However, at present, this protein is not recovered and considerable quantities of blood and cruor are rejected, leaving a noble product to be lost, and in addition, causing heavy pollution during discharges. This loss is all the more detrimental since globin has extremely interesting technological properties such as very pronounced foaming and emulsifying powers (which are superior to those of egg albumin and plasma which are products currently widely used to fill these functions).

This lack of recovery and recovery of blood derivatives stems from a practical inability to prepare, under satisfactory economic conditions, a product containing globin, which is substantially white in order to be usable as an additive. Indeed, at the present time essentially four types of process are known which make it possible to eliminate the coloring of hemoglobin, but all these processes have drawbacks which, in practice, make recovery unprofitable or very poorly profitable.

A first type of process consists in carrying out the bleaching by means of concentrated hydrogen peroxide. The specific drawbacks of this process are as follows: dangers arising from the handling of concentrated hydrogen peroxide and from the presence of traces of hydrogen peroxide in the final product, strong denaturation of the protein which loses its technological properties, implementation extremely delicate industrial process on large quantities due to the formation of considerable volumes of foam, high cost of the process due to the high cost of the basic product used.

Another type of process consists in treating hemoglobin by means of ethanol which causes the formation of heme aggregates or derivatives and allows the separation of globin. However, the quantities of ethanol required are very high and the process must be carried out at low temperature (below -5 ° C.). Therefore, the cost of its industrial implementation is high and does not make it possible to make the recovery of globins from the blood profitable.

Another type of process, widely used in the medical field for analyzes or research, consists in placing the hemoglobin in acetonic acid medium; under these conditions the globin precipitated in white flakes can be separated from the heme which remains in solution. This process, which is very effective in ensuring complete separation, however has the serious drawback of forcing the handling of acetone which is a very dangerous product, with significant risks of toxic residues in the globin obtained, making its consumption dangerous. This process, acceptable for analyzes where the separated globin is not consumed, is no longer so for obtaining a globin intended for food. In addition, it leads to acetone consumption extremely high, which must then be regenerated by distillation.

A final type of process consists in hydrolyzing hemoglobin, chemically in a highly concentrated acid medium, or enzymatically in the presence of protease, in order to split the hemoglobin into various constituents: one of them contains the heme which, insoluble, can be separated. Reference may be made, for example, to patents AU 449710 or FR 2379988 to illustrate the techniques of chemical hydrolysis. However, during hydrolysis, globin is itself broken down into very small peptides (chain of 3 to 4 amino acid residues) or even amino acids, which makes it lose its technological properties and gives it a bitter taste, making it difficult to use as a food additive. Furthermore, the hydrolysis by chemical means is carried out under restrictive operating conditions (very long heating time of the order of 24 hours, presence of an acid medium with a concentration at least equal to 6N, pressure generally greater than the atmospheric pressure).

It is essential to note that, in the case of the hydrolysis techniques mentioned above, the products obtained are not the proteins themselves but devalued fractions of them and the competent professionals have accepted that it was not possible, by simple techniques of hydrolysis in an acid medium, to separate proteins from prosthetic groups and to isolate them without breaking the peptide bonds. In addition, experiments have shown that the products obtained by these techniques had a very pronounced red-brown color.

Under these conditions, the shortcomings summarized above in the conventional procedures mean that enormous quantities of blood from slaughterhouses are currently rejected without any recovery; in some cases, the yellow plasma is recovered but the cruor which is the remaining red product, containing hemoglobin, is lost. The present invention proposes to to overcome the flaws and shortcomings of conventional methods of hemoglobin treatment in order to allow chemical and physical separation of globin without general breakdown of the peptide bonds thereof and under very economical conditions of implementation.

More generally, the invention proposes to indicate a treatment method making it possible to separate one or more proteins from their prosthetic group or groups without appreciably altering the protein and without risk of any trace of toxic or dangerous product in it. this.

An objective of the invention is in particular to provide a tasteless and white protein, retaining all of its properties and in particular its technological properties.

Another objective is to rigorously avoid any manipulation of dangerous or toxic products and to allow obtaining a protein strictly free of toxic residues. Another objective is to provide a treatment using very inexpensive raw materials, benefiting from a simple and easy implementation at low temperature and, consequently, capable of being developed on an industrial level at an extremely low cost. . To this end, the treatment method according to the invention consists of:

- In a first step, to make a dilute acid solution, consisting of the protein substance, water and acid, having a pH between 0.5 and 5, so as to cause the rupture of most of the bonds proteins / prosthetic groups without very sensitive hydrolysis of the peptide bonds of said proteins, to form a precipitate Pl containing the major part of said prosthetic group or groups and to preferentially keep in solution the protein or proteins of the substance, - and, in a second step, to separate, by a liquid / solid physical separation process, the solid phase PI obtained from the liquid phase L1.

Experiments have shown that, very unexpectedly, it forms in an acidic aqueous medium diluted to the aforementioned pH and in the absence of addition of any auxiliary product, a precipitate preferably containing the prosthetic groups, which can be easily separated by any known route (centrifugation, filtration, decantation, etc.) to obtain a liquid phase very poor in prosthetic groups. These experiments have in particular brought to light the essential fact and completely contrary to the ideas of professionals consisting in the ability, at low acid dilution, to break the major part of the protein / prosthetic group bonds without generalized breakdown of the peptide bonds and then to separate these proteins through a simple physical / liquid separation process. Uninsulated white proteins are thus isolated, with a molecular weight of the order of 8,000 to 12,000, whereas, in prior hydrolysis techniques, which use acid concentrations in the reaction medium from 10 to 50 times higher (patent AU 449710 and patent FR 2379988), the products obtained are small peptides with molecular weights less than 700, poorly isolated from prosthetic groups

(red / brown color). Thus, in the case of hemoglobin, by implementing the method of the invention the liquid phase obtained / is weakly colored and contains for the most part globin of high molecular weight.

It should be noted that the initial prosthetic groups are generally transformed during the treatment to provide derived groups but without the protein itself having undergone significant modifications (that is to say capable of appreciably altering its properties). The preferred operating conditions of the process according to the invention are as follows:

- use of sulfuric acid,

- concentration by weight of the protein substance in the solution approximately between 5 and 30 grams per liter,

- solution heated to a temperature approximately between 80º C and 100º C for a period of time between one hour and three hours before proceeding to the separation step. These operating conditions are gentle and make it possible to increase the quantity of protein separated and to reduce the residual proportion of prosthetic group in the liquid phase. They also entail the essential and remarkable advantage of causing aggregation or bonding of the particles of the precipitate through the gelation of a certain proportion of the protein substance. The following liquid / solid separation operation is greatly facilitated and can be carried out in a few minutes, for example by simple filtration through a common filter paper or by centrifugation or decantation techniques.

Furthermore, it has been observed that, during the first step, the addition to the solution of a certain amount of precipitate formed during a previous treatment considerably promotes the formation of the PI precipitate and makes it possible to increase the quantity of precipitate formed during a given period of time and to obtain, after separation, a liquid phase depleted in prosthetic groups.

Furthermore, it has been demonstrated that the separate solid phase PI which contains the major part of the prosthetic groups also contains protein substance which has not been split with respect to said groups. The process can be limited to a single cycle with a view to recovering from the liquid phase L1 only a portion of the proteins practically free from the prosthetic groupings which generate them. It is also possible to recover a larger quantity of proteins by subjecting, at the end of the second step, the solid phase PI to the following additional treatment: dilution of this phase in water in order to allow the passage into solution of part of the proteins contained therein and separation of the new liquid phase L3 and of the new solid phase P3 after a determined period of time in order to recover an additional quantity of proteins in said liquid phase, these operations possibly , if applicable, be reproduced. Furthermore, according to another mode of implementation which can be combined with the previous one, the process can be adapted to obtain a more concentrated solution of proteins in order in particular to reduce the drying costs. In this embodiment, the liquid phase L1 obtained after separation of the precipitate PI is brought to a pH corresponding to the minimum solubility of the proteins concerned; for example, for globin, this pH is substantially between 7 and 7.5 and corresponds to a very reduced solubility of globin. A new fluffy P2 precipitate is formed, which is rich in protein and low in salt. This precipitate is then separated from the new liquid phase L2 in order to obtain concentrated proteins, containing little salt.

This latter mode of implementation is in particular very advantageous when the protein substance has been the subject of a prior conservation (or other) treatment with toxic salts: it then allows natural elimination of most of these salts.

The method of the invention applies in particular in the case of colored protein substances (in particular hemoglobin or chlorophyll and substances derived therefrom) to obtain from these substantially white proteins. Applications of the hemoglobin and chlorophyll process and substances derived therefrom are illustrated by the examples described below. Example 1 - The starting body treated is cruor (which consists of the blood centrifugation pellet from which the supernatant plasma has been separated).

The cruor treated in this example comes from beef blood collected on a mixture of sulfite and citrate.

An acidic aqueous medium is prepared by mixing a strong mineral acid with water; in the example,

26 cm of 12 N HCL were added to 3.6 liters of water.

400 cm ³ of cruor are then poured into this medium and the solution, the pH of which is homogenized, is homogenized around 2.1. We wait 24 hours during which we note the formation of a blackish precipitate PI which settles at the bottom of the container; the remaining liquid solution L1 is light brown in color.

After 24 hours, filtration is carried out on conventional filter paper. In this example, the blackish precipitate PI is eliminated and the collected liquid phase L1 is analyzed; it contains a weight concentration of around 15 g / liter of globin.

This liquid phase L1 is then subjected to an atomization treatment which makes it possible to obtain approximately 50 g of white powder, slightly tinted brown. The initial quantity of treated cruor contained approximately 90 g of globin bound to heme in the form of hemoglobin: taking into account the salts present, it was thus recovered in a single cycle and by an extremely simple process of implementation, approximately the half of the globin initially present. Example 2 - The two steps of the process of Example 1 are repeated to obtain a liquid phase L1 of the same nature after elimination of the precipitate PI.

1.7 liters of 0.1 N sodium hydroxide are poured into this liquid phase (the volume of which is approximately 3.5 liters) so as to bring the pH to a value equal to 7. A white fluffy precipitate is immediately formed rosé P2 which is left to settle.

The supernatant liquid L2 phase is eliminated; this phase contains the major part, on the one hand, of the salts formed by implementing the process during the acid and base additions, on the other hand, of the salts present or added in the initial cruor (in particular sulfites and citrates).

The flaky precipitate P2 which is therefore rich in globin and poor in salt is dissolved in a non-neutral aqueous medium, so as to form a solution of pH of the order of 5 to 5.5, containing 30 g of precipitate per liter of solution. Under these conditions, it is found that all of the precipitate has gone into solution. We then perform a treatment of hydration by atomization and a very slightly pink white powder is obtained; his analysis shows that this powder contains 82% globin. EXAMPLE 3 The two stages of the process of Example 1 are repeated, but the solid phase PI obtained after filtration is preserved, in order to subject it to an additional treatment allowing this to recover part of the globins contained in it. . This solid phase PI is diluted three times in volume in water to obtain a new solid phase P3 and a new liquid phase L3. This new P3 phase is allowed to settle for 48 hours and the supernatant L3 liquid phase is removed. This liquid phase, which contains part of the proteins remaining in the PI phase, is light yellow in color.

This phase is atomized to transform it into powder. A perfectly white powder is obtained containing by weight a proportion of approximately 80% of globin.

Example 4 - An acidic aqueous medium is prepared by mixing 65 cm ³ of 12 N HCL in 9 liters of water and poured into this medium 1 liter of cruor obtained from blood collected on phosphate and sodium chloride. The solution, the pH of which is about 2.2, is homogenized.

We wait about 12 hours during which we note the formation of a blackish precipitate P'l which settles; the remaining liquid solution L'l is light brown in color

Centrifugation is carried out and approximately 9 liters of L'l supernatant are recovered. This solution is passed over an activated carbon column; it is found that the solution obtained L "l is notably clearer. The activated carbon therefore makes it possible to eliminate part of the prosthetic groups which were still contained in the solution L'l.

Then added to the solution L "l, 4.3 liters of 0.1 N sodium hydroxide: there is the formation of a precipitate P'2 of whitish color. This precipitate is sou put in sunlight for two days; it can be seen that its whiteness is accentuated, which demonstrates an effect of degradation of the prosthetic groups by light.

The process continues by dissolving the precipitate in an acid medium and atomizing the new liquid phase obtained. This produces an extremely white powder containing approximately 85% globin.

Example 5 - An acidic aqueous medium is prepared by mixing 78 cm of 12 N HCL in 8.4 liters of water and poured into this medium 1.2 liters of cruor obtained from blood collected on citrate and sulfite. The solution, the pH of which is about 2.2, is homogenized.

The solution is brought to 90 ° C for 15 minutes and there is the formation of a blackish precipitate which settles during cooling; the remaining liquid solution is pale yellow in color.

The total volume remaining after cooling is 6.4 liters. By filtration, 3.8 liters of pale yellow solution are recovered, containing approximately 48.5 g / liter of globins, or by weight 57% of the initial hemoglobin.

The powder obtained after dehydration is white in color (without any pinkish tinge). The temperature is therefore a favorable factor which makes it possible to considerably reduce the reaction time, while increasing the yield and the quality of whiteness obtained.

Example 6 - This example relates to the separation of proteins and prosthetic groups contained in a blue algae residue of the spirulina type. These algae contain the following protein substances: chlorophyll 1, carotenoid, xanthophyll, phycobilin (phycocyanin and phycoerythrin).

An acidic aqueous medium is prepared by mixing 3.5 cm of 12 N HCl in 500 cm ³ of water and 30 g of powdered spirulina residues are poured into this medium, obtained after rough separation of the blue protein substance. The solution, the pH of which is around 3, is homogenized (part of the powder remains in the undissolved state).

The solution is brought to 90 ° C for 50 minutes and there is the formation of a dark green precipitate which settles during cooling; the remaining liquid solution is light yellow in color. The total volume remaining after cooling is 400 cm ³ . By centrifugation, we recover

250 cm ³ of light yellow solution, containing approximately 14.1 g / liter of the aforementioned proteins, or 14% of the initial proteins. The powder obtained after dehydration is white (without any colored shade).

Example 7 a) A solution is prepared by mixing 9.5 liters of water with 19 cm of H ₂ SO ₄ 36 N and 400 cm of cruor with 32.25% dry extract. The pH of the solution is thus 1.70 and the concentration in moles of H ₃ O ⁺ is 0.063 moles / liter of solution. b) The solution is heated to 98 ° C for

2 hours. As the reaction progresses, solid aggregates with the appearance of a gel are formed. Thus the solid particles have a large size which greatly facilitates the liquid / solid separation of step C. c) At the end of these two hours, a liquid / solid separation of the solution is carried out by filtration on ordinary filter paper or on cotton canvas or on a washable synthetic canvas. Filtration is very fast: 10 to 20 minutes. d) 7 liters of very light, sterile or practically sterile pale yellow permeate are thus collected and

3 liters of insoluble dark brown retentate. The pH of the permeate is 1.90. Its dry extract is 1.13%. The quantity of product recovered in the permeate is therefore equal to 61% of the quantity of product present in the initial solution. The pH of the retentate is 2.10. Its dry extract is 1.66%. The quantity of product recovered in the retentate is therefore equal to 39% of the quantity of product present in the initial solution. e) By adding 280 cm of 2N sodium hydroxide the pH of the permeate is brought to 9.00 (which corresponds substantially to the pH of egg white). The permeate can be used as it is (in liquid form) or preconcentrated (by evaporation or ultrafiltration) then dehydrated (by atomization) to be transformed into powder. f) The white protein powder of the cruor can then be used in particular in the preparation of a large number of pastry products because of its very high foaming power. A non-exhaustive list of these pastries is as follows: meringues, madeleines, macaroons, sponge cakes, shortbread, spoon cookie, chocolate mousse, soufflés ...

100 ml of dry extract egg white equal to 12% gives after whipping, for 3 to 5 minutes, about 450 ml of foam.

100 ml of dry extract cruor protein equal to 0.8% gives, under the same threshing conditions, approximately 1,500 ml of foam.

The firmness and resistance of the foam over time from cruor proteins is comparable to that of egg white. Firmness and hold are very significantly enhanced by the presence of sugars (sucrose for example) or salts (NaCl for example).

This improvement in the consistency of the foam is very interesting in the pastry industry where threshing for the preparation of the foam is often carried out in the presence of sugar.

The table below makes it possible to compare the quantities of foaming agent necessary (egg white or white cruor proteins) for the preparation of 1 kg of certain pastries.

This therefore means in concrete terms that, for example, 30 to 40 times more egg white powder than white cruor protein powder is required to make madeleines. g) The aminogram of the white cruor protein powder obtained is as follows: Amino acids (in g per 100 g of protein):

Aspartic acid 8.29

Threonine 4.29

Serine 5.40

Glutamic acid 6.85

Proline 3.60

Wisteria 5.11

Alanine 10.85

Cystine 0.64

Valine 8.01

Methionine 1.16

Isoleucine 0.10

Leucine 12.65

Tyrosine 3.02

Phenylalanine 6.44

Lysine 10.04

Histidine 8.99

Arginine 3.02

Tryptophan 1.57

Example 8 - The first 4 steps (a, b, c, d) of Example 7 are repeated.

The initial cruor is composed of 80 to has 90% of hemoglobins containing themselves 4% of prosthetic groups.

At the end of steps a, b, c, d, the permeate is very light pale yellow and contains practically no more prosthetic groups. On the contrary, the retentate is a mixture of prosthetic groups of origin and their derivatives with a remainder of protein substances. In this example we are interested in this retentate which can be used, among other things, for animal feed.

The foaming power of the retentate is high, although lower than that of the permeate. 100 ml of retentate of dry extract 1.66% of pH adjusted to 9.00 with 2 N sodium hydroxide gives after threshing 350 ml of light brown foam and very firm consistency.

The retentate which contains around 80% protein in the dry extract can also be used as a source of protein, lysine (10% of the total amino acids) and easily assimilated iron (around 0.3% of the dry extract) for animal feed. The insolubility in water of this retentate can in particular make it interesting for feeding fish. The aminogram of the retentate is as follows: Amino acids (in g per 100 g of protein):

Aspartic acid 8.30

Threonine 4.29 Serine 5.40

Glutamic acid 6.85

Proline 3.60

Glycine 5, H

Alanine 10.80 Cystine 0.64 Phenylalanine 6.44

Valine 8.01 Lysine 10.03

Methionine 1.16 Histidine 8.99

Isoleucine 0.10 Arginine 3.02

Leucine 12.65 Tryptophan 1.57 Tyrosine 3.02 Example 9 - The first two steps a, b of Example 7 are repeated.

At the end of these two hours, the pH of the solution is adjusted between 4 and 5 using 2N sodium hydroxide.

The liquid / solid separation is then normally carried out. The permeate obtained is pale yellow, has a pH between 4.2 and 5.2 and is perfectly soluble. If the filtration is carried out under aseptic conditions, this permeate is sterile. Its dry extract is around 1%.

The permeate can be used liquid or dehydrated.

The foaming power of this permeate is approximately the same as that of the permeate of Example 7.

The solubility, the pH, the sterility, the foaming power, the protein content of the product give it an important interest in cosmetology. This liquid permeate or after dehydration can also be used in cold meats because of its foaming and emulsifying power and its pH.

Example 10 - The solution is prepared by mixing 2,000 liters of water with 2.1 1 H ₂ SO ₄ 36 N and 100 liters of cruor with 32.50% dry extract. The solution has a pH of 2.79.

This solution is heated for 1.5 hours at 98 ° C.

At the end of this 1 hour 30 minutes, the liquid / solid separation is carried out under the conditions defined in step c) of Example 7.

1,400 liters of very light pale yellow permeate are collected. The pH of the permeate is 3.20. The permeate has a dry extract of 0.99%. The amount of product recovered in the permeate is therefore equal to 43% of the amount of product present in the initial solution.

Claims

1) Method for treating a protein substance composed of at least one protein and at least one prosthetic group, with a view to at least partially separating said protein (s) from said prosthetic group (s), characterized in that it consists : - in a first step, to make dilute acid a solution / consisting of the protein substance, water and acid, having a pH between 0.5 and 5, so as to cause the rupture of most of the protein / prosthetic group bonds without generalized rupture of the peptide bonds of said proteins, and to form a PI precipitate containing the major part of said prosthetic group (s) and to preferentially keep the protein or proteins of the substance,

- And, in a second step, to separate, by a liquid / solid physical separation process, the solid phase PI obtained from the liquid phase L1. 2) A treatment method according to claim 1, characterized in that the solution is made so that the weight concentration of the protein substance is approximately between 5 and 30 grams per liter of solution.

3) Treatment method according to one of claims 1 or 2, characterized in that the solution containing the protein substance is brought to a temperature approximately between 80 ° C and 100 ° C for a period of time approximately between 1 and 3 hours before proceeding to the separation step.

4) Treatment process according to one of claims 1, 2 or 3, characterized in that the acid used to make the above solution is sulfuric acid.

5) Treatment method according to one of claims 1, 2, 3 or 4, characterized in that, during the first step, is added to the solution an amount of solid phase P'l obtained during a previous treatment to catalyze precipitation rolling during said first stage.

6) Treatment method according to one of claims 1, 2, 3, 4 or 5, characterized in that the liquid phase L1 obtained at the end of the second step is brought to a pH corresponding to the minimum protein solubility, in with a view to forming a precipitate P2 rich in proteins and poor in salt, said precipitate P2 then being separated from the new liquid phase L2 in order to obtain concentrated proteins.

7) Treatment process according to claim 6, characterized in that the precipitate P2, rich in proteins and poor in salt, is dissolved at least partially in a non-neutral aqueous medium, the two phases are optionally mixed and undergo the dehydration treatment of the liquid or mixture obtained, with a view to transforming it into powder.

8) Treatment method according to one of claims 1, 2 or 3, 4, 5 or 6 or 7, characterized in that, at the end of the second step, the solid phase obtained PI is diluted in water, with a view to dissolving part of the proteins contained in said solid phase, the new liquid phase L3 being separated from the new solid phase P3, with a view to recovering an additional quantity of proteins, this operation being if necessary reproduced.

9) Treatment method according to one of the preceding claims, in which the second step consists in carrying out one of the following separation operations: centrifugation, filtration or decantation.

10) Method of treatment according to one of the preceding claims of a protein substance containing at least one protein and at least one colored prosthetic group, in order to obtain substantially white proteins, isolated with respect to an insoluble colored residue.

11) The method of claim 10 applied to cruor containing as proteinaceous hemoglobin. 12) Method according to claim 11 characterized in that the solution is brought to a temperature of 98 to 99 ° C for a time between 1 h 30 and 2 h 30.

13) Method according to claim 10 applied to a vegetable protein substance containing chlorophyll.